Utilizing a post-translational assay that I recently developed for signal peptidase, I propose to purify and characterize this enzyme which is responsible for removing the transient hydrophobic signal peptide region of presecretory and premembrane proteins, as described by the Signal Hypothesis. Purification will utilize the techniques of ion exchange chromatography, hydrophobic chromatography, preparative isoelectric focusing and affinity chromatograpy. Characterization will include determination of its molecular weight, subunit structure and amino acid composition, as well as elucidation of its disposition with respect to the phospholipid bilayer of the membrane. Enzymatic characterization will include the identification of inhibitors and an investigation of its role in the process of segregation. Its kinetic properties will be investigated with synthetic signal peptide substrates. Its ubiquity will be examined immunologically. One product of the signal peptidase reaction is the authentic secretory or membrane protein, the other is the signal peptide. As part of this comprehensive study of signal peptidase, the metabolic fate of signal peptide will be determined. Other elements of the segregation apparatus (e.g., the ribosome receptor proteins) will be identified and purified through their association with signal peptidase and their recently elucidated sensitivity to protein modification reagents. The function of these other elements will be determined both functionally and immunologically. Dysfunction of the segregation apparatus or mutations in the signal region of specific secretory or membrane proteins would undoubtedly be deleterious; hence, the proposed research has potential medical relevance for such diseases as diabetes and cancer.